BILE ACID METABOLISM AND PLASMA CHOLESTEROL REGULATION

肝胆固醇7-α-羟基酶催化初始和速率- 胆固醇转化为胆固醇的主要途径中的限制步骤 胆盐。因此，7-α-羟基酶在这两个过程中都起着核心作用。 维持全身的类固醇平衡，其次，在 调节血清脂蛋白浓度。肝脏7-α- 对胆汁的反应在转录水平上调节羟基酶 盐、脂肪酸，在某些物种中还含有胆固醇；然而， 分子机制是由这些主要的生理调节因子 改变转录的7-α-羟基酶基因是完全 未知。这在很大程度上是由于缺乏体外培养或细胞 复制所观察到的主要监管形式的文化体系 在活体内。此外，含有启动子/报告基因的转基因大鼠 构建长达4kb的7-α-羟基酶5-侧翼DNA 表现出极低水平的转基因表达 受胆汁或胆固醇调节，表明其他序列 比在4 kb的5‘-侧翼DNA中所需的 在体内正常表达和调节。该计划的总体目标 拟议的研究是为了了解7-α转录是如何- 羟基酶基因受胆盐和脂肪酸的调节 确定胆固醇上调的机制 7-α-羟基酶基因在某些物种中的转录(大鼠和 老鼠)，但在其他(仓鼠)中不存在。使用细胞核和核提取液 来自饲喂胆盐、胆盐隔离剂、脂肪酸或 胆固醇，我们建议对大鼠和仓鼠进行系统的评估 从DNase I超敏部位开始的7-α-羟基酶基因座 DNase识别重要调控序列的作图 I足迹和甲基化干扰研究以识别核 因子结合位点。选定的足迹将使用凝胶进行分析 迁移率变化分析和竞争对手寡核苷酸识别已知 转录因子。以这种方式确定的潜在增强将 克隆在7-α-羟基酶近端启动子(或a 异源启动子)和增强子活性的检测以及 在体内对生理调节剂的反应能力 腺病毒介导的基因转移。在不太可能的情况下， 介导对主要生理反应的序列 使用体内瞬时转染法无法确定调节子 以上研究概述，我们将使用转基因动物来定位 负责调节的序列。转基因小鼠最初将是 用大鼠7-α-羟基酶基因的P1克隆产生。 然后将在指导下进行删除突变分析 DNase I超敏位点定位结果。这些研究将 提供有关转录调控的关键信息 体内的7-α-羟基酶基因，并可能为 制定新的战略，以加强对 将胆固醇转化为胆盐，降低心血管风险。

Hepatic cholesterol 7-alpha-hydroxylase catalyzes the initial and rate- limiting step in the major pathway where by cholesterol is converted to bile salts. As such, 7-alpha-hydroxylase plays a central role both in the maintenance of whole body sterol balance and, secondarily, in the regulation of serum lipoprotein concentrations. Hepatic 7-alpha- hydroxylase is regulated at the transcriptional level in response to bile salts, fatty acids and, in some species, cholesterol; however, the molecular mechanisms where by these major physiological regulators alter transcription of the 7-alpha-hydroxylase gene are completely unknown. This is due, in large part, to the lack of in vitro or cell culture systems that reproduce the major forms of regulation observed in vivo. Moreover, transgenic rats containing promoter/reporter constructs up to 4 kb of 7-alpha-hydroxylase 5 -flanking DNA manifested very low levels of transgene expression that were minimally regulated by bile slats or cholesterol indicating that sequences other than those present in the 4 kb of 5'-flanking DNA are required for normal expression and regulation in vivo. The overall goal of the proposed research is to understand how transcription of the 7-alpha- hydroxylase gene is regulated by bile salts and fatty acids and to determine the mechanism whereby cholesterol up regulates transcription of the 7-alpha-hydroxylase gene in some species (rats and mice) but not in others (hamsters). Using nuclei and nuclear extracts from animals fed bile salts, bile salt sequestrants, fatty acids or cholesterol, we propose a systematic evaluation of the rat and hamster 7 -alpha-hydroxylase gene loci starting with DNase I hypersensitive site mapping to identify important regulatory sequences followed by DNase I footprinting and methylation interference studies to identify nuclear factor binding sites. Selected footprints will be analyzed using gel mobility shift assays and competitor oligonucleotides to identify known transcription factors. Potential enhances identified in this manner will be cloned next to the 7-alpha-hydroxylase proximal promoter (or a heterologous promoter) and testing for enhancer activity and the ability to confer responsiveness to physiologic regulators in vivo using adenovirus-mediated gene transfer. In the unlikely event that the sequences mediating responsiveness to the major physiological regulators cannot be identified using the in vivo transient transfection studies outline above, we will use transgenic animals to map the sequences responsible for regulation. Transgenic mice will initially be generated using P1 clone of the rat 7-alpha-hydroxylase gene. Deletional mutagenesis analysis will then be carried out guided by the results of DNase I hypersensitive site mapping. These studies will provide critical information regarding the transcriptional regulation of the 7-alpha-hydroxylase gene in vivo and may open the door for the development of novel strategies aimed at enhancing the conversion of cholesterol to bile salts and reducing cardiovascular risk.